Most human genes produce multiple distinct mRNA and protein isoforms through alternative splicing (AS). AS can produce protein isoforms that often have distinct or even antagonistic biological functions, is often tissue- specific, and contributes to various diseases. Our approach to the long-term goal of understanding the regulation and evolution of AS in mammals is organized around the following specific aims: SA1. Understand the evolution of alternative mRNA isoforms in mammals and the nature of genomic changes that alter splicing patterns. SA2. To develop analytical methods for quantitative inference of mRNA isoform abundance and translational activity from RNA-Seq and ribosome footprinting (Ribo-Seq) data, and to develop models to understand quantitative changes in organism- and tissue-specific regulation of mRNA isoforms. SA3. To construct a splicing regulatory network of mouse embryonic stem cells (mESCs). Our lab is generating RNA-Seq data spanning 10 diverse tissues from each of 5 vertebrate species - rhesus macaque, mouse, rat, cow and chicken. We will apply state-of-the art tools for RNA-Seq-based genome annotation to these and available human data, to annotate the transcriptomes of these organisms. These data will be used to trace the evolutionary histories of the exon-intron structures and splicing patterns of mammalian genes. We will extend a Bayesian mixture model we have developed to estimate levels of alternative isoforms from RNA-Seq data in several ways, including integrating with transcriptome annotation tools and extending to Ribo-Seq data, and we will develop models to quantitatively predict alternative isoform abundance. Finally, we will determine auto- and cross-regulatory relationships between 50 major splicing regulatory factors expressed in mESCs in order to construct a network model of splicing regulation in this fundamental cell type, using RNAi and overexpression coupled with RNA-Seq and Ribo-Seq. Together, these studies will provide a comprehensive basis for understanding alternative mRNA isoform regulation in mammals. PUBLIC HEALTH RELEVANCE: This project will provide comprehensive resources, tools and concepts for understanding the different protein forms produced by human genes, including their evolutionary histories and regulation. These resources, tools and concepts will aid in understanding mammalian development and the pathogenic mechanisms of a variety of diseases related to misregulation of alternative splicing, including myotonic dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, retinitis pigmentosa and cancer.